Backbone modified oligonucleotide analogs and preparation thereof through radical coupling

ABSTRACT

Methods for preparing oligonucleotide analogs which have improved nuclease resistance and improved cellular uptake are provided. In preferred embodiments, the methods involve radical coupling of 3&#39;- and 5&#39;-substituted or 5&#39;- and 3&#39;-subutituted nucleosidic synthons.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is division of patent application Ser. No.08/300,072, filed on Sep. 2, 1994 (now U.S. Pat. No. 5,618,704), whichis a continuation of patent application Ser. No. 08/040,933, filed onMar. 31, 1993 (now abandoned). Ser. No. 08/040,933 is acontinuation-in-part of application Ser. No. 07/903,160, filed on Jun.24, 1992 (now abandoned), and application PCT/US92/04294, filed on May21, 1992 (now abandoned). Application Ser. No. 07/903,160 is acontinuation-in-part of application Ser. No. 07/703,619, filed on May21, 1991 (now U.S. Pat. No. 5,378,825), application Ser. No. 07/566,836,filed on Aug. 13, 1990 (now U.S. Pat. No. 5,223,618), application Ser.No. 07/558,663, filed on Jul. 27, 1990 (now U.S. Pat. No. 5,138,045),and application PCT/US92/04294. Application PCT/US92/04294 is acontinuation-in-part of application Ser. No. 07/703,619, which is acontinuation-in-part of application Ser. No. 07/566,836 and applicationSer. No. 07/558,663. This patent application also is related to thesubject matter disclosed and claimed in application Ser. No. 08/039,979,filed on Mar. 30, 1993 (now abandoned), application Ser. No. 08/039,846,filed on Mar. 30, 1993 (now abandoned), application Ser. No. 08/040,526,filed on Mar. 31, 1993 (now U.S. Pat. No. 5,489,677), and applicationSer. No. 08/040,903, filed on Mar. 31, 1993 (now U.S. Pat. No.5,386,023). Each of these patent applications are assigned to theassignee of this patent application and are incorporated by referenceherein.

FIELD OF THE INVENTION

This invention relates to the design, synthesis and application ofnuclease resistant oligonucleotide analogs which are useful fortherapeutics, diagnostics and as research reagents. Oligonucleotideanalogs are provided having modified linkages replacing thephosphorodiester bonds that serve as inter-sugar linkages in wild typenucleic acids. Such analogs are resistant to nuclease degradation andare capable of modulating the activity of DNA and RNA. Methods forsynthesizing these oligonucleotide analogs and for modulating theproduction of proteins also are provided.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are effected by proteins. Proteins, either actingdirectly or through their enzymatic functions, contribute in majorproportion to many diseases in animals and man.

Classical therapeutics generally has focused upon interactions withproteins in an effort to moderate their disease causing or diseasepotentiating functions. Recently, however, attempts have been made tomoderate the production of proteins by interactions with the molecules(i.e., intracellular RNA) that direct their synthesis. Theseinteractions have involved hybridization of complementary "antisense"oligonucleotides or certain analogs thereof to RNA. Hybridization is thesequence-specific hydrogen bonding of oligonucleotides oroligonucleotide analogs to RNA or to single stranded DNA. By interferingwith the production of proteins, it has been hoped to effect therapeuticresults with maximum effect and minimal side effects.

The pharmacological activity of antisense oligonucleotides andoligonucleotide analogs, like other therapeutics, depends on a number offactors that influence the effective concentration of these agents atspecific intracellular targets. One important factor foroligonucleotides is the stability of the species in the presence ofnucleases. It is unlikely that unmodified oligonucleotides will beuseful therapeutic agents because they are rapidly degraded bynucleases. Modification of oligonucleotides to render them resistant tonucleases therefore is greatly desired.

Modification of oligonucleotides to enhance nuclease resistancegenerally has taken place on the phosphorus atom of the sugar-phosphatebackbone. Phosphorothioates, methyl phosphonates, phosphoramidates andphosphorotriesters have been reported to confer various levels ofnuclease resistance. Phosphate-modified oligonucleotides, however,generally have suffered from inferior hybridization properties. See,e.g., Cohen, J. S., ed. Oligonucleotides: Antisense Inhibitors of GeneExpression, (CRC Press, Inc., Boca Raton Fla., 1989).

Another key factor is the ability of antisense compounds to traverse theplasma membrane of specific cells involved in the disease process.Cellular membranes consist of lipid-protein bilayers that are freelypermeable to small, nonionic, lipophilic compounds and are inherentlyimpermeable to most natural metabolites and therapeutic agents. See,e.g., Wilson, Ann. Rev. Biochem. 1978, 47, 933. The biological andantiviral effects of natural and modified oligonucleotides in culturedmammalian cells have been well documented. It appears that these agentscan penetrate membranes to reach their intracellular targets. Uptake ofantisense compounds into a variety of mammalian cells, including HL-60,Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells has beenstudied using natural oligonucleotides and certain nuclease resistantanalogs, such as alkyl triesters and methyl phosphonates. See, e.g.,Miller, et al., Biochemistry 1977, 16, 1988; Marcus-Sekura, et al., Nuc.Acids Res. 1987, 15, 5749; and Loke, et al., Top. Microbiol. Immunol.1988, 141, 282.

Often, modified oligonucleotides and oligonucleotide analogs areinternalized less readily than their natural counterparts. As a result,the activity of many previously available antisense oligonucleotides hasnot been sufficient for practical therapeutic, research or diagnosticpurposes. Two other serious deficiencies of prior art compounds designedfor antisense therapeutics are inferior hybridization to intracellularRNA and the lack of a defined chemical or enzyme-mediated event toterminate essential RNA functions.

Modifications to enhance the effectiveness of the antisenseoligonucleotides and overcome these problems have taken many forms.These modifications include base ring modifications, sugar moietymodifications and sugar-phosphate backbone modifications. Priorsugar-phosphate backbone modifications, particularly on the phosphorusatom, have effected various levels of resistance to nucleases. However,while the ability of an antisense oligonucleotide to bind to specificDNA or RNA with fidelity is fundamental to antisense methodology,modified phosphorus oligonucleotides have generally suffered frominferior hybridization properties.

Replacement of the phosphorus atom has been an alternative approach inattempting to avoid the problems associated with modification on thepro-chiral phosphate moiety. For example, Matteucci, Tetrahedron Letters1990, 31, 2385 disclosed the replacement of the phosphorus atom with amethylene group. However, this replacement yielded unstable compoundswith nonuniform insertion of formacetal linkages throughout theirbackbones. Cormier, et al., Nucleic Acids Research 1988, 16, 4583,disclosed replacement of phosphorus with a diisopropylsilyl moiety toyield homopolymers having poor solubility and hybridization properties.Stirchak, et al., Journal of organic Chemistry 1987, 52, 4202 disclosedreplacement of phosphorus linkages by short homopolymers containingcarbamate or morpholino linkages to yield compounds having poorsolubility and hybridization properties. Mazur, et al., Tetrahedron1984, 40, 3949, disclosed replacement of a phosphorus linkage with aphosphonic linkage yielded only a homotrimer molecule. Goodchild,Bioconjugate Chemistry 1990, 1, 165, disclosed ester linkages that areenzymatically degraded by esterases and, therefore, are not suitable forantisense applications.

The limitations of available methods for modification of the phosphorusbackbone have led to a continuing and long felt need for othermodifications which provide resistance to nucleases and satisfactoryhybridization properties for antisense oligonucleotide diagnostics andtherapeutics.

OBJECTS OF THE INVENTION

It is an object of the invention to provide oligonucleotide analogs fordiagnostic, research, and therapeutic use.

It is a further object of the invention to provide oligonucleotideanalogs capable of forming duplex or triplex structures with, forexample, DNA.

It is a further object to provide oligonucleotide analogs havingenhanced cellular uptake.

Another object of the invention is to provide oligonucleotide analogshaving greater efficacy than unmodified antisense oligonucleotides.

It is yet another object of the invention to provide methods forsynthesis and use of oligonucleotide analogs.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the present specification and theappended claims.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that mimic and/ormodulate the activity of wild-type nucleic acids. In general, thecompounds contain a selected nucleoside sequence which is specificallyhybridizable with a targeted nucleoside sequence of single stranded ordouble stranded DNA or RNA. At least a portion of the compounds of heinvention has structure I: ##STR1## wherein: L₁ --L₂ --L₃ --L₄ is CH₂--R_(A) --NR₁ --CH₂, CH₂ --NR₁ --R_(A) --CH₂, R_(A) --NR₁ --CH₂ --CH₂,or NR₁ --R_(A) --CH₂ --CH₂ ;

R_(A) is O or NR₂ ;

R₁ and R₂ are the same or different and are H; alkyl or substitutedalkyl having 1 to about 10 carbon atoms; alkenyl or substituted alkenyl2 to about 10 carbon atoms; alkynyl or substituted alkynyl having 2 toabout 10 carbon atoms; alkaryl, substituted alkaryl, aralkyl, orsubstituted aralkyl having 7 to about 14 carbon atoms; alicyclic;heterocyclic; a reporter molecule; an RNA cleaving group; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of anoligonucleotide;

B_(X) is a nucleosidic base;

n is an integer greater than 0;

Q is O, S, CH₂, CHF or CF₂ ;

X is H, OH, C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl, an RNA cleaving group, a group for improving thepharmacokinetic properties of an ligonucleotide, or a group forimproving the pharmacodynamic roperties of an oligonucleotide.

The compounds of the invention generally are prepared by couplingpreselected 3'-functionalized and 4'-functionalized nucleosides and/oroligonucleotides under conditions effective to form the above-noted L₁--L₂ --L₃ --L₄ linkages. In preferred embodiments, the linkages areformed by coupling synthons having structures II and III: ##STR2##wherein: Z₁ and Y₂ are selected such that:

(i) Z₁ is R_(B) and Y₂ is CH₂ --R_(A) --N═CH₂ ; or

(ii) Z₁ is CH₂ --R_(B) and Y₂ is R_(A) --N═CH₂ ; or

(iii) Z₁ is CH₂ --R_(A) --N═CH₂ and Y₂ is R_(B) or

(iv) Z₁ is R_(A) --N═CH₂ and Y₂ is CH₂ --R_(B) ;

where the synthon bearing said R_(B) group being a donor synthon and thesynthon bearing said N═CH₂ group being an acceptor synthon;

R_(S) is a radical generating group selected from I, OC(O)O--C₆ H₅,OC(O)S--C₆ H₅, Se--C₆ H₅, O--C(S)O--C₆ F₅, O--C(S)O--C₆ Cl₅,O--C(S)O--(2,4,6--C₆ Cl₃), Br, NO₂, Cl, C(S)S-Me, C(S)O--(p-CH₄ F),bis-dimethylglyoximato-pyridine cobalt (i.e., C(dmgH)₂ PY) OC(S)C₆ H₅,OC(S)SCH,₃ OC(S)-imidazole, and C(O)O-pyridin-2-thione;

Y₁ and Z₂ are, independently, H, hydroxyl, aminomethyl, hydrazinomethyl,hydroxymethyl, C-formyl, phthalimidohydroxy- methyl, aryl-substitutedimidazolidino, aminohydroxylmethyl, ortho-methylaminobenzenethio,methylphosphonate, methyl-alkylphosphonate, a nucleoside, a nucleotide,an oligonucleotide, an oligonucleoside, or a hydroxyl-protected oramine-protected derivative thereof;

B_(X1) and B_(X2) are, independently, nucleosidic bases;

Q₁ and Q₂ are, independently, O, S, CH₂, CHF or CF₂ ; and

X₁ and X₂ are, independently, H, OH, C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl,S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂,NP₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino or substituted silyl, an RNA cleaving group, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide.

In general, the coupling reaction entails generating a carbon-centeredradical appended at either a 3' or 5' position of one synthon, andreacting that radical with a radical acceptor group appended to a 5' or3' position of the other synthon. This reaction scheme preferably isrepeated a number of times to produce a desired nucleosidic sequence.

DETAILED DESCRIPTION OF THE INVENTION

The term "nucleoside" as used in connection with this invention refersto a unit made up of a heterocyclic base and its sugar. The term"nucleotide" refers to a nucleoside having a phosphate group on its 3'or 5' sugar hydroxyl group. Thus nucleosides, unlike nucleotides, haveno phosphate group. "oligonucleotide" refers to a plurality of joinednucleotide units formed in a specific sequence from naturally occurringbases and pentofuranosyl groups joined through a sugar group by nativephosphodiester bonds. This term refers to both naturally occurring andsynthetic species formed from naturally occurring subunits.

The compounds of the invention generally can be viewed as"oligonucleotide analogs", that is, compounds which function likeoligonucleotides but which have non-naturally occurring portions.Oligonucleotide analogs can have altered sugar moieties, altered basemoieties or altered inter-sugar linkages. For the purposes of thisinvention, an oligonucleotide analog having non-phosphodiester bonds,i.e., an altered inter-sugar linkage, is considered to be an"oligonucleoside." The term "oligonucleoside" thus refers to a pluralityof nucleoside units joined by linking groups other than nativephosphodiester linking groups. The term "oligomers" is intended toencompass oligonucleotides, oligonucleotide analogs or oligonucleosides.Thus, in speaking of "oligomers" reference is made to a series ofnucleosides or nucleoside analogs that are joined via either naturalphosphodiester bonds or other linkages, including the four atom linkersof this invention. Although the linkage generally is from the 3' carbonof one nucleoside to the 5' carbon of a second nucleoside, the term"oligomer" can also include other linkages such as 2'-5' linkages.

Oligonucleotide analogs also can include other modifications consistentwith the spirit of this invention, particularly modifications thatincrease nuclease resistance. For example, when the sugar portion of anucleoside or nucleotide is replaced by a carbocyclic moiety, it is nolonger a sugar. Moreover, when other substitutions, such a substitutionfor the inter-sugar phosphorodiester linkage are made, the resultingmaterial is no longer a true nucleic acid species. All such compoundsare considered to be analogs. Throughout this specification, referenceto the sugar portion of a nucleic acid species shall be understood torefer to either a true sugar or to a species taking the structural placeof the sugar of wild type nucleic acids. Moreover, reference tointer-sugar linkages shall be taken to include moieties serving to jointhe sugar or sugar analog portions in the fashion of wild type nucleicacids.

This invention concerns modified oligonucleotides, i.e., oligonucleotideanalogs or oligonucleosides, and methods for effecting themodifications. These modified oligonucleotides and oligonucleotideanalogs exhibit increased stability relative to their naturallyoccurring counterparts. Extracellular and intracellular nucleasesgenerally do not recognize and therefore do not bind to thebackbone-modified compounds of the invention. In addition, the neutralor positively charged backbones of the present invention can be takeninto cells by simple passive transport rather than by complicatedprotein-mediated processes. Another advantage of the invention is thatthe lack of a negatively charged backbone facilitates sequence specificbinding of the oligonucleotide analogs or oligonucleosides to targetedRNA, which has a negatively charged backbone and will repel similarlycharged oligonucleotides. Still another advantage of the presentinvention is it presents sites for attaching functional groups thatinitiate cleavage of targeted RNA.

The modified internucleoside linkages of this invention preferablyreplace naturally-occurring phosphodiester-5'-methylene linkages withfour atom linking groups to confer nuclease resistance and enhancedcellular uptake to the resulting compound. Preferred linkages havestructure CH₂ --R_(A) --NR₁ --CH₂, CH₂ --NR₁ --R_(A) --CH₂, R_(A) --NR₁--CH₂ --CH₂ or NR₁ --R_(A) --CH₂ --CH₂ where R_(A) is O or NR₂.

Generally, these linkages are prepared by functionalizing the sugarmoieties of two nucleosides which ultimately are to be adjacent to oneanother in the selected sequence. In a 4' to 3' sense, an "upstream"synthon such as structure II is modified at its terminal 3' site, whilea "downstream" synthon such as structure III is modified at its terminal4' site. More specifically, the invention provides efficient,stereoselective syntheses of oligonucleosides via intermolecular radicaladdition. The radical addition reaction can be divided in two steps. Thefirst step involves generation of an initial radical, which undergoesthe desired reaction. The second step involves removal of the radicalfrom the reaction before the occurrence of an intervening, undesiredreaction such as cross coupling. In certain embodiments, the linkages ofthe invention are prepared by providing donor synthons having structureIIa or IIIa: ##STR3## wherein Z_(1a), and Y_(2a) have structure CH₂--R_(B) or R_(B) where R_(B) is a radical generating group, generating aradical centered at Z_(1a) or Y_(2a), and then forming a 3'-5' linkageby reacting radical-bearing donor synthons IIA and IIIa, respectively,with acceptor synthons IIIb and IIb: ##STR4## wherein either Z_(1b) andY_(2b) have structure R_(A) N═CH₂ or CH₂ --R_(A) N═CH₂ where R_(A) is Oor NR₂.

B_(X1) and B_(X2) can be nucleosidic bases selected from adenine,guanine, uracil, thymine, cytosine, 2-aminoadenosine or5-methylcytosine, although other non-naturally occurring species can beemployed to provide stable duplex or triplex formation with, forexample, DNA. Representative bases are disclosed in U.S. Pat. No.3,687,808 (Merigan, et al.), which is incorporated herein by reference.

Q₁ and Q₂ can be S, CH₂, CHF, CF₂ or, preferably, O. See, e.g., Secrist,et al., Abstract 21, Synthesis and Biological Activity of4'-Thionucleosides, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992.

X₁ and X₂ are, independently, H, OH, C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl,S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino or substituted silyl, an RNA cleaving group, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide. It is preferred that X is H or OH, or, alternatively F,O-alkyl or O-alkenyl, especially where Q is O. Preferred alkyl andalkenyl groups have from 1 to about 10 carbon atoms.

Y₁ and Z₂ can be H, hydroxyl, aminomethyl, hydrazinomethyl,hydroxymethyl, C-formyl, phthalimidohydroxymethyl, aryl-substitutedimidazolidino, aminohydroxylmethyl, ortho-methylaminobenzenethio,methylphosphonate, methyl-alkylphosphonate, a nucleoside, a nucleotide,an oligonucleotide, an oligonucleoside, or a hydroxyl-protected oramine-protected derivative thereof. Preferably, Y₁ is a protectedhydroxymethyl group or a nucleoside or oligonucleoside attached by, forexample, a phosphodiester-5'-methylene linkage or some other four atomlinking group, and Z₂ is a protected hydroxyl group or a nucleoside oroligonucleoside attached by, for example, a phosphodiester-3'-hydroxyllinkage or some other four atom linking group.

It is preferred that the oligonucleotide analogs of the inventioncomprise from about 5 to about 50 subunits 35 having the given structure(i.e., n=5-50). While each subunit of the oligonucleotide analogs canhave repetitive structure I, such need not be the case. For example, thesubunits can have alternating or more random structures.

The methods of the invention generally involve "nonchain" processes. Innonchain processes, radicals are generated by stoichiometric bondhomolysis and quenched by selective radical-radical coupling. It hasbeen found that bis(trimethylstannyl)benzopinacolate andbis(tributyl-stannyl)benzopinacolate (see Comprehensive OrganicSynthesis: Ed. by B. M. Trost & J. Fleming, Vol. 4, pp 760)--persistentradicals--can be used to enhance the radical-radical coupling and reducecross-coupling. It will be recognized that a persistent radical is onethat does not react with itself at a diffusion-controlled rate.Hillgartner, et al., Liebigs. Ann. Chem. 1975, 586, disclosed that onthermolysis (about 80° C.) pinacolate undergoes homolytic cleavage togive the suspected persistent radical (Ph₂ C.sup.· OSnMe₃), which staysin equilibrium with benzophenone and the trimethylstannyl radical (Me₃Sn•). It is believed that the Me₃ Sn• radical abstracts iodine fromradical precursors such as 3'-deoxy-3'-iodo nucleosides or5'-deoxy-5'-iodo nucleoside derivatives to give 3' or 5' nucleosideradicals. The nucleoside radicals then add to, for example, oxime etheracceptors such as 3'- or 5'-deoxy-3' or 5'-methyleneamino-oxy nucleosidederivatives to give a dimeric nucleoside containing a dephosphointernucleoside linkage.

The concentration of the persistent radical is an important variable inthese reactions because at high concentrations the initial radical canbe trapped by coupling prior to addition, and at low concentrations theadduct radical can begin to telomerize. It is believed that a 3 molarequivalent excess of pinacolate provides satisfactory results for suchcouplings. The efficiency of radical reactions are highly dependent onthe concentration of the reagents in an appropriate solvent. Preferably,the solvent contains, for example, benzene, dichlorobenzene,t-butylbenzene, t-butyl alcohol, water, acetic acid, chloroform, carbontetrachloride, and mixtures thereof. The solvent should contain acombined concentration of about 0.1 to about 0.4 moles/liter of radicalprecursor and acceptor, preferably about 0.1 to about 0.2 moles/liter,more preferably about 0.2 moles/liter. It has been found that bestresults are obtained using benzene solutions containing about 0.2moles/liter of radical precursor and acceptor.

As exemplified in Scheme I, chain elongation in a 5' to 3' sense can beachieved generally by refluxing a 0.2-0.4 molar solution of5'-deoxy-5'-iodo-3'-0-phthalimido nucleoside 102 (R'=hydroxyl protectinggroup, R"=phthalimido), 3,-deoxy-3'-methylene amino-oxy-5'-protectednucleoside 101, and bis(trimethylstannyl)-benzopinacolate in benzeneunder argon for 8 h to yield dimeric nucleoside 103 (L₁ --L₂ --L₃ --L₄=O--NH--CH₂ --CH₂) in 35% yield after urification by silica gelchromatography. This dimer was ethylated following standard proceduresas for instance utilizing aqueous formaldehyde (20% solution) to furnishN-alkylated 104 (L₂ =N--CH₃) in high yield. Further hydrazinolysis ofand formulation of the product will furnish 3'-oxime ether 105(R"=N═CH₂), which is ready for another round of radical coupling. Thus,coupling this dimer with bifunctional nucleoside 102 will provide trimer106 (L_(1a) --L_(2a) --L_(3a) --L_(4a) =O--N(H)--CH₂ --CH₂, n=1). In asimilar manner, trimer 106 can undergo another round of coupling tofurnish a tetrameric nucleoside. Repetitive coupling of this type willprovide an oligomer of desired length. Chain elongation can beterminated at any time during the described method. For example,coupling of dimer ether 105 with a 5'-deoxy-5'-iodo-3'-protectednucleoside will furnish trimer 107 (L_(2a) =N--CH₃), which could beN-methylated (L₂ =N--CH₃) and deblocked at its 3' and 5' ends to yielddeprotected trimer 108 (R'=R"=H). ##STR5##

As exemplified in Scheme II, chain elongation in a 3' to 5' sense can beachieved generally by refluxing a concentrated (0.2 to 0.3 molar)solution of 3'-deoxy-3'-iodo-5'-O-phthalimido nucleoside 109,5'-deoxy-5' [(methyleneamino)oxy]-3'-protected nucleoside 110, andbis(trimethylstannyl)benzopinacolate in benzene under argon for 8 h toyield dimeric nucleoside 111 (L₁ --L₂ L₃ --L₄ =CH₂ --NH--O--CH₂,R'=hydroxyl protecting group, R"=phthalimido) in 45% yield afterpurification by silica gel chromatography. Dimer 111 was methylatedfollowing standard procedures to furnish N-alkylated 112 (L₂ =N--CH₃) ingood yield. Subsequently, hydrazinolysis of 112, followed by formylationof the product will furnish 113 (R'=N═CH₂). Dimer 113 can undergoanother round of radical coupling with 109 to yield trimeric nucleoside114 (L_(1a) --L_(2a) --L_(3a) --L_(4a) =O--N(H)--CH₂ --CH₂, n=1). Thelatter compound could be N-methylated to yield 115 (L_(2A) =N--CH₃). Arepetitive set of reactions such as hydrazinolysis, formylation, andcoupling would result in an oligomer of desired length containingmodified backbones. Chain elongation can be terminated at any point bycoupling with 3'-deoxy-3'-iodo-5'-O-protected nucleoside 109. Forexample, the latter compound will couple with 113, and the product onmethylation (L₂ =N--CH₃) and deblocking will furnish trimeric nucleoside116 (R'=R"=H).

A more random type of elongation-can be effected by deblocking andiodinating nucleoside 104 selectively at its 3' end to producenucleoside 117a (R'=O-blocking group, R"=I). Coupling of 117a with 110via 3'-elongation furnishes trimeric nucleoside 118a (L₂ =N--CH₃ andL_(2a) =NH). Methylation and complete deblocking of 118a provides 118b(R'=R"=OH and L₁ =L₂ =N--CH₃). Alternatively, deblocking and iodinatingnucleoside 112 selectively at its 5' end produces nucleoside 119a (R'=I,R"=O-blocking group). Coupling of 119a with 101 via 5'-elongationfurnishes trimeric nucleoside 118a. Methylation and complete deblockingprovides 118b. ##STR6##

A wide range of achiral and neutral oligonucleosides containing mixedbackbones can be prepared by these "random" methods. Backbone complexitycan be enhanced further by selectively incorporating phosphodiesterlinkages to, for example, increase water solubility. (See, e.g., Example27). Also, coupling 5'-O-dimethyoxytritylated dimeric nucleoside 117b(R'=ODMTr, R"=OH) or trimeric nucleoside 118c (R"=ODMTr, R"=OH, L₂=N--CH₃ and L_(2a) =NH) to CPG via a succinyl linker (see, e.g., NucleicAcids Research 1990, 18, 3813) provides CPG-bound compounds that can beused as 3'-terminal units for automated synthesis. The placement of suchdimeric or trimeric oligonucleosides at the 3'-end of an antisenseoligomer will result in the protection of the antisense molecules fromattack by 3'-exonucleases typically found in human serum.

In summary, the chain elongation methods of the invention requireessentially two types of nucleoside building blocks. The first is amonofunctional nucleoside with one reactive group for coupling at 3' or5' end and an appropriate blocking group at the remaining end. Thesecond is a bifunctional nucleoside which has a reactive coupling groupat 3' or 5 end and a protected reactive coupling group at the other end.

The methods of the invention can be modified for use with eithersolution-phase or solid-phases techniques. For example, the compounds ofthe invention can be synthesized using controlled pore glass (CPG)supports and standard nucleic acid synthesizing machines such as AppliedBiosystems Inc. 380B and 394 and Milligen/Biosearch 7500 and 8800s. Eachnew nucleoside is attached either by manual manipulation or by automatedtechniques.

A wide variety of protecting groups can be employed in the methods ofthe invention. See, e.g., Beaucage, et al., Tetrahedron 1992, 12, 2223.In general, protecting groups render chemical functionality inert tospecific reaction conditions, and can be appended to and removed fromsuch functionality in a molecule without substantially damaging theremainder of the molecule. Representative hydroxyl protecting groupsinclude t-butyldimethylsilyl (TBDMSi), t-butyldiphenylsilyl (TBDPSi),dimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and other hydroxylprotecting groups as outlined in the above-noted Beaucage reference.

Scheme III illustrates certain abbreviations used for blocking groups inother of the schemes. Scheme III further shows the synthesis of3'-O-amino and 3'-O-methyleneamino nucleosides via a Mitsunobu reactionutilizing N-hydroxylphthalimide and methylhydrazine to generate an--O--NH₂ moiety on a sugar hydroxyl. The --O--NH₂ group can then bederivatized to a --O-methyleneamino moiety. These reactions areexemplified in Examples 5, 6, 7, 9 and 18.

The reactions of Examples 5, 6, 7 and 9 represent an improved synthesisof 3'-O-NH₂ nucleosides. In forming --O--NH₂ moieties on sugars, it istheoretically possible to displace a leaving group, such as a tosylgroup, with hydroxylamine. However, Files, et al., J. Am. Chem. Soc.1992, 14, 1493, have shown that such a displacement leads to apreponderance of --NHOH moieties and not to the desired --O--NH₂moieties. Further, the reaction sequence of Examples 5, 6, 7 and 9represents an improved synthesis compared to that illustrated inEuropean Patent Application 0381335. The synthetic pathway of thatpatent application requires the use of a xylo nucleoside as the staringmaterial. Xylo nucleosides are less readily obtainable than theribonucleoside utilized in Examples 5, 6, 7 and 9.

Scheme VIII illustrates a synthetic scheme utilized to prepare dimers,trimers, and other, higher-order oligonucleosides having homogenouslinkages between nucleosides. In this scheme, nucleosides 10 and 12 arelinked to form an iminomethylene linkage as exemplified in Example 11.Advantageous use of the alkylating-5' terminus deblocking step of SchemeVI is effected to remove the blocking group at the 5' terminus of thedimeric oligonucleoside 14, as in Example 12. Using the iodinationreaction of Scheme IV, the dimer then is converted to a 5' terminus iodointermediate, as in Example 14. A further 35 3'-O-methyleneaminonucleosidic unit 10 then can be added to the dimer to form a trimer, asin Example 15, followed by deblocking and alkylation, as in Example 16.This reaction sequence can be repeated any number of times to form ahigher order oligonucleoside. The oligonucleoside is deblocked at the 3'terminus, as is exemplified for the dimer in Example 13 or the tetramerin Example 17.

Scheme IX illustrates a radical reaction that forms a linkage having apendant hydroxyl moiety. This is exemplified in Example 21. The pendantOH group can be oxidized to an ═O using Moffatt oxidization conditions.Alternatively, the pendant OH moiety can be cyclized to the nitrogenatom of the linkage to form either a five or a six membered heterocyclicring. The formation of a linkage incorporating a six atom ring isexemplified in Example 22. A five atom ring would be formed utilizingcondition analogous to those of Neumeyer, et al., J. Org. Chem. 1973,38, 2291, to add phosgene in the presence of a base such astriethylamine or diethylphenylamine in toluene at a temperature of about60 to about 80° C. ##STR7##

The compounds of this invention can be used in diagnostics,therapeutics, and as research reagents and kits. For therapeutic use theoligonucleotide analog is administered to an animal suffering from adisease modulated by some protein. It is preferred to administer topatients suspected of suffering from such a disease an amount ofoligonucleotide analog that is effective to reduce the symptomology ofthat disease. One skilled in the art can determine optimum dosages andtreatment schedules for such treatment regimens.

It is preferred that the RNA or DNA portion which is to be modulated bepreselected to comprise that portion of DNA or RNA which codes for theprotein whose formation or activity is to be modulated. The targetingportion of the composition to be employed is, thus, selected to becomplementary to the preselected portion of DNA or RNA, that is to be anantisense oligonucleotide for that portion.

In accordance with one preferred embodiment of this invention, thecompounds of the invention hybridize to HIV mRNA encoding the tatprotein, or to the TAR region of HIV mRNA. In another preferredembodiment, the compounds mimic the secondary structure of the TARregion of HIV mRNA, and by doing so bind the tat protein. Otherpreferred compounds complementary sequences for herpes, papilloma andother viruses.

It is generally preferred to administer the therapeutic agents inaccordance with this invention internally such as orally, intravenously,or intramuscularly. Other forms of administration, such astransdermally, topically, or intra-lesionally may also be useful.Inclusion in suppositories may also be useful. Use of pharmacologicallyacceptable carriers is also preferred for some embodiments.

This invention is also directed to methods for the selective binding ofRNA for research and diagnostic purposes. Such selective, strong bindingis accomplished by interacting such RNA or DNA with compositions of theinvention which are resistant to degradative nucleases and whichhybridize more strongly and with greater fidelity than knownoligonucleotides or oligonucleotide analogs.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting,wherein parts and percents are by weight unless otherwise indicated. ForNMR analysis of dimers and other higher oligonucleosides, monomericunits are numbered (e.g., T₁, T₂) from the 5' terminus towards the 3'terminus nucleoside. Thus, the 5' nucleoside of a T-T dimer is T₁ andthe 3' nucleoside is T₂.

General Procedures

Radical Addition Reaction

A suspension of radical precursor (1-3 eq.), radical acceptor (1 eq.),persistent radical (3 eq.) in benzene (0.2-0.4 mol. solution) wascarefully degassed under vacuum (water aspirator) and flushed with argon(3-20 times). The reaction mixture was heated at 80-85° C. for 6-12 hunder argon while stirring. The suspension dissolves to give a clearsolution in about 1-2 h. The reaction mixture may change the colorduring the course of heating but remains clear all along. Completion ofthe reaction was judged by the disappearance of radical precursor(detected by TLC) and formation of a polar product. The reaction mixturethen was cooled to room temperature and diluted with ether (five timesthe original volume). The fine suspension was loaded onto a prepackedsilica gel column (30 g of silica per gram of product) and eluted withhexanes (100%) until most of the UV absorbing impurities were removed.The column then was eluted with a hexanes-ether gradient in which theconcentration of ether gradually increased to effect a 10% increase inthe polarity of the solvent. Elution with ether furnished the desiredproduct as homogeneous material. Pooling and evaporation of appropriatefractions generally gave 40-55% yield of the desired dimeric nucleoside.

N-Alkylation of Backbone

To a stirred solution of oligonucleoside (1 eq.; containing CH₂--O--NH--CH₂ linkages) in glacial acetic acid was added an aqueoussolution of HCHO (3-5 eq.; >99% HCHO) in one portion under argon. Theclear solution was stirred for 5-15 min. at room temperature until nomore starting material was detected by TLC. At this point dry NaBH₃ CN(3-6 eq.) was added in 3-6 portions under argon at room temperatureunder a well ventilated fume hood. Care must be taken to cool thereaction mixture if it is over 5 mmol. scale. After addition, thereaction mixture was stirred for 1-2 h at room temperature (untilevolution of gas ceases). Completion of the reaction was detected byTLC, which indicated formation of a higher product, compared to thestarting material. The reaction mixture was concentrated under vacuumand the residue purified by short column chromatography. Elution with aCH₂ Cl₂ →MeOH gradient (increasing polarity to 10%) furnished thedesired product as homogeneous material.

This procedure was applicable to a variety of substituted aldehydeswhich formed Schiff bases with the amino group of the linker. Subsequentreduction gave selective alkylation of the backbone. Heterocyclic orexocyclic amines of the purine/pyrimidine were not affected by thismethod.

Composition Analysis of Modified oligomers

Incorporation of oligonucleoside containing backbones of the inventioninto antisense molecules was proved by enzymatic tandem hydrolysis ofmodified oligomers using snake-venom phosphodiesterase followed byalkaline phosphatase. In all cases, the identity of dimeric nucleosideswas proven by addition of synthetic sample and comparison on HPLCprofile. The integration of the peaks of HPLC analysis demonstrated thecorrect gross composition of the modified oligomer.

EXAMPLE 1

Synthesis of 5'-O-Phthalimido Nucleosides

5'-O-Phthalimidothynidine

To a stirred solution of thymidine (24.22 g, 0.1 mol),N-hydroxyphthalimide (21.75 g, 0.13 mol), triphenylphosphine (34 g, 0.13mol) in dry DMF (400 ml) was added diisopropylazodicarboxylate (30 ml,0.15 mol) over a period of 3 h at 0° C. After complete addition thereaction mixture was warmed up to room temperature and stirred for 12 h.The solution was concentrated under vacuum (0.1 mm., <40° C.) to furnishan orange-red residue. The residual gum was washed several times withEt₂ O and washing were discarded. The semi-solid residue was suspendedin EtOH (500 ml) and heated (90° C.) to dissolve the product. On cooling30.98 g (80%) of 5'-Q-phthalimidothymidine was collected in 3-crops aswhite crystalline material, mp 233-235° C. (decomp.); ¹ H NMR (DMSO-d₆)δ 11.29 (s, 1, NH), 7.85 (m , 4, ArH), 7.58 (s, 1, C₆ H), 6.20 (t, 1,H_(1'), J_(1'),2 '=7.8 Hz, J_(1'),2" =6.5 Hz), 5.48 (d, 1, OH₃,), 4.36(m, 3, H_(4'),5',5"), 4.08 (m, 1, H_(3")), 2.09-2.13 (m, 2, H_(2'),2"),and 1.79 (s, 3, CH₃). Anal. Calcd. for C₁₈ H₁₇ O₇ N₃.sup.· 0.7 H₂ O: C,54.05; H, 4.64; N, 10.51. Found: C, 53.81; H, 4.25; N, 10.39.

2'-deozy-5'-O-phthalimidouridine

An analogous reaction on 2'-deoxyuridine gave the corresponding2'-deoxy-5'-O-phthalimidouridine; mp 241-242°C.

2'-deozy-5'-O-phthalimidocytidine

An analogous reaction on 2'-deoxycytidine gave the corresponding2'-deoxy-5'-O-phthalimidocytidine in 40% yield.

2'-deozy-5'-O-phthalimidoadenosine

An analogous reaction on 2'-deoxyadenosine gave the corresponding2'-deoxy-5'-O-phthalimidoadenosine in 55% yield.

2'-deoxy-5'-O-phthalimidoguanosine

An analogous reaction on 2'-deoxyguanosine gave the corresponding2'-deoxy-5'-O-phthalimidoguanosine in 25% yield.

EXAMPLE 2

Synthesis of 5'-O-phthalimido-3'-O-(t-butyldiphenyl-silyl)thymidine and2'-deozy-5'-O-phthalisido-3'-O-(t-butyldiphenylsilyl)uridine

3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothysidine

A mixture of 5'-O-phthalimidothymidine (8.54 g, 22 mmol),t-butyldiphenylsilylchloride (6.9 ml, 26.5 mmol), imidazole (3.9 g, 57.3mmol) and dry DMF (130 ml) was stirred at room temperature for 16 hunder argon. The reaction mixture was poured into ice-water (600 ml) andthe solution was extracted with CH₂ Cl₂ (2×400 ml). The organic layerwas washed with water (2×250 ml) and dried (MgSO₄). The CH₂ Cl₂ layerwas concentrated to furnish a gummy residue which on purification bysilica gel chromatography (eluted with EtOAc:Hexanes; 1:1, v/v)furnished 12.65 g (92%) of3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothymidine as crystallinematerial (mp 172-173.5° C.). ¹ H NMR (DMSO-d₆) δ 11.31 (s, 1, NH), 7.83(m, 4, ArH), 7.59 (m, 4, TBDPhH), 7.51 (s, 1, C₆ H), 7.37-7.45 (m, 6,TBDPhH), 6.30 (dd, 1, H_(1'), J_(1'),2' =8.8 Hz, J_(1'),2" =5.6 Hz),4.55 (m, 1, H_(4')), 4.15 (m, 1, H_(3')), 3.94-4.04 (m, 2, H_(5'),5")2.06-2.13 (m, 2, H_(2'),2"), 1.97 (s, 3, CH₃), 1.03 (s, 9, C(CH₃)₃).Anal. Calcd. for C₃₄ H₃₅ O₇ N₃ Si: C, 65.26; H, 5.64; N, 6.71. Found: C,65.00; H, 5.60; N, 6.42.

3'-O-(t-butyldiphenylsilyl)-2'-deoy-5'-O-phthalimidouridine

An analogous reaction of 2'-deoxy-5'-O-phthalimidouridine will give thecorresponding3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidouridine.

3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidocytidine

An analogous reaction of 2'-deoxy-5'-O-phthalimidocytidine gave thecorresponding3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidocytidine in 65%yield.

3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidoadenosine

An analogous reaction of 2'-deoxy-5'-O-phthalimidoadenosine gave thecorresponding3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidoadenosine in 70%yield.

3'-O-(t-butyldiphanyluilyl)-2'-deoxy-5'-O-phthalimidoguanosine

An analogous reaction of 2'-deoxy-5'-O-phthalimidoguanosine gave thecorresponding3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidoguanosine in 65%yield.

EXAMPLE 3

Synthesis of 5'-O-amino nucleoside

5'-O-amino-3'-O-(t-butyldiphenylsilyl)thysidine

To a stirred solution of3'-O-(t-butyldiphenyl-silyl)-5'-O-phthalimidothymidine (10 g, 16 mmol)in dry CH₂ Cl₂ (100 ml) was added methylhydrazine (1.3 ml, 24 mmol)under argon at room temperature and solution stirred for 12 h. Thesolution was cooled (0° C.) and filtered. The white residue was washedwith CH₂ Cl₂ (2×25 ml) and combined filtrates were evaporated to furnishgummy residue. The residue on purification by silica gel columnchromatography (elution with CH₂ Cl₂ :MeOH, 98:2, v/v) furnished 7.03 g(89%) of 5'-O-amino-3'-O-(t-butyldiphenylsilyl)thymidine thatcrystallized from CH₂ Cl₂ /MeOH mp 141-143° C. ¹ H NMR (DMSO-d₆) δ 11.29(s, 1, NH), 7.42-7.62 (m, 11, TBDPhH, C₆ H), 6.25 (dd, 1, H_(1'),J_(1'),2' =8.4 Hz, J_(1'),2" =6.3 Hz), 6.02 (s, 2, NH₂), 4.35 (m, 1,H_(4')), 4.04 (m, 1, H_(3')), 3.34-3.51 (m, 2, H_(5'),5"), 2.04 (m, 2,H_(2'), 2"), 1.73 (s, 3, CH₃), 1.03 (s, 9, C(CH₃)₃). Anal. Calcd. forC₂₆ H₃₃ O₅ N₃ Si: C, 63.00; H, 6.71; N, 8.48. Found: C, 62.85; H, 6.67;N, 8.32.

EXAMPLE 4

Synthesis of methylated [3'--CH₂ --N(CH₃)--O--CH₂ --5'] linkedoligonucleoside

3'-De(ozyphosphinico)-3'-[methylene(methyl-imino)]thymidylyl-(3'→5')thymidine

To a stirred solution of3'-de(oxyphosphinico)-3'-(methyleneimino)thymidylyl-(3'→5')-3'-O-(t-butyldi-phenylsilyl)thymidinedimer (0.99 g, 1 mmol) in glacial AcOH (10 ml) was added aqueous HCHO(20%, 3 ml). The solution was stirred for 5 min. at room temperature andto this was added NaBH₃ CN (0.19 g, 3 mmol) in 3-portions under argon atroom temperature. The addition of NaBH₃ CN (0.19 g) was repeated oncemore and solution was further stirred for 1 h. The reaction mixture wasconcentrated to furnish crude3'-de(oxyphosphinico)-3'-[methylene(methylimino)]-thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidinedimer, which on deblocking (nBu₄ NF/THF, HCl/MeOH) furnished the titlecompound,3'-de(oxyphosphinico)-3'-[methylene-(methylimino)]thymidylyl-(3'→5')thymidine, (0.44 g, 87%) as white solids. The3'-de(oxyphosphinico)-3'-[methylene-(methylimino)]thymidylyl-(3'→5')thymidine dimer was further purified by preparative HPLC furnishing ananalytically pure sample. ¹ H NMR (DMSO-d₆) δ 11.30 and 11.24 (2s, 2,2NH), 7.82 and 7.50 (2s, 2, 2C₆ H), 6.15 (pseudo t, 1, T2 H_(1'),J_(1'),2' =6.3 Hz, J_(1'),2" =7.3 Hz), 6.00 (pseudo t, 1, T1 H_(1'),J_(1'),2' =4.2 Hz, J_(1'),2" =6.1 Hz), 5.31 (m, 1, T2 OH), 5.08 (m, 1,T1, OH), 4.17 (m, 1, T2 H_(3')), 3.88 (m, 1, T2 H_(4')), 3.57-3.83 (m,5, T1 T2 H₄═,5", T1 H_(4')), 2.69 (m, 2, T1 H_(3")), 2.57 (s, 3,N--CH₃), 2.50 (m, 1, T1 H_(3')), 2.05-2.14 (m, 4, T1 T2 H_(2'),2"), 1.79and 1.76 (2s, 6, 2 CH₃). MS FAB: M/z 510 (M+H)⁺. Anal. Calcd. for C₂₃H₃₁ N₅ O₉.sup.· H₂ O: C, 50.09; H, 6.31; N, 13.28. Found: C, 50.05; H,6.21, N, 13.08.

EXAMPLE 5

5'-O-(t-Butyldiaothylsilyl)-3'-O-Phthalimidothynidine, 2

To a solution of 5'-O-t-butyldimethylsilylthymidine [1, 21.36 g, 60mmol, prepared according to the procedure of Nair, et al., Org. Prep.Procedures Int. 1990, 22, 57 in dry THF (750 ml)], triphenylphosphine(17.28 g, 66 mmol) and N-hydroxyphthalimide (10.74 g, 66 mmol) wereadded. The solution was cooled to 0° C. and diisopropylazodicarboxylate(15.15 g, 75 mmol) was added dropwise over a period of 3 hr whilestirring under nitrogen. The reaction mixture was then stirred at roomtemperature for 12 hr. The solution was evaporated and the residue wasdissolved in CH₂ Cl₂ (750 ml), extracted with sat. NaHCO₃ (200 ml), andwater (200 ml), dried (MgSO₄), filtered and concentrated to furnishyellow oily residue. Silica gel column chromatography (100% hexanes, andthen hexanes:Et₂ O gradient to 90% Et₂ O) of the residue gave compound 2as a colorless glass (18.68 g, 62%); ¹ H NMR (CDCl₃) δ 0.05 [2s, 6,(CH₃)₂ ], 0.91 [s, 9, (CH₃)₃ ], 2.0 (s, 3, CH₃), 2.5-2.65 (m, 2, 2'CH₂),4.05-4.2 (m, 2, 5'CH₂), 4.25-4.35 (m, 1, 4'H), 5.0 (m, 1, 3'H), 6.15 (m,1, 1'H), 8.6 (br s, 1, NH), and aromatic protons. Anal. Calcd. for C₂₄H₃₁ N₃ O₇ Si: C, 57.46; H, 6.23; N, 8.37. found: C, 57.20; H, 6.26; N,8.27.

EXAMPLE 6

3'-O-Amino-5'-O-(t-Butyldimethylsilyl)thysidine, 3

Cold methylhydrazine (1.6 ml, 30 mmol) was added to a stirred solutionof 5'-O-(t-butyldimethylsilyl)-3'-O-phthalimidothymidine (2, 4.6 g, 9.18mmol) in dry CH₂ Cl₂ (60 ml) at 5-10° C. After 10 minutes whiteprecipitation of 1,2-dihydro-4-hydroxy-2-methyl-1-oxophthalizineoccurred. The suspension was stirred at room temperature for 1 h. Thesuspension was filtered and precipitate washed with CH₂ Cl₂ (2×20 ml).The combined filtrates were concentrated and the residue purified bysilica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(100:0→97:3, v/v) furnished the title compound (3.40 g, 100%) as whitesolid. Crystallization from CH₂ Cl₁ gave white needles, m.p. 171° C.; ¹H NMR (CDCl₃) δ 0.05 [s, 6, (CH₃)₂ ], 0.90 [s, 9, (CH₃)₃ ], 2.22-2.58(2m, 2, 2'CH₂), 3.9-4.08 (m, 3, 5'CH₂, and 3'H) 4.30 (m, 1, 4'H) 5.5 (brs, 2, NH₂) 6.2 (m, 1, 1'H) 7.45 (s, 1, C₆ H) 8.9 (br s, 1, NH). Anal.Calcd. for C₁₆ H₂₉ N₃ O₅ Si: C, 51.72; H, 7.87; N, 11.32. found: C,51.87, H, 7.81; N, 11.32.

EXAMPLE 7

3'-O-Aminothymidine, 4

3'-O-Amino-(t-butyldimethylsilyl)thymidine was deblocked with (Bu)₄NF/THF in standard way to furnish compound 4 (72%). Crystallized fromether/hexanes/ethanol as fine needles, mp 81° C. ¹ H NMR (Me₂ SO-d₆) δ1.78 (s, 3, CH₃), 2.17 and 2.45 (2m, 2, 2'CH₂), 3.70 (m, 2, 5'CH₂), 3.88(m, 1, 4'H), 4.16 (m, 1, 3'H), 4.8 (br s, 1, 5'OH), 6.05 (dd, 1, 1'H),6.2 (br s, 2 NH₂), 7.48 (s, 1, C₆ H), and 11.24 (br s, 1, NH). Anal.Calcd. for C₁₀ H₁₅ N₃ O₅ : C, 46.69; H, 5.87; N, 16.33; found: C, 46.55;H, 5.91; N, 16.21.

EXAMPLE 8

3'-O-Dephosphinico-3'-O-(Methylimino)thyidylyl-(3'→5')-5,-Deoxythymidine,9

Step 1

3'-O-Amino-5'-O-(t-butyldimethylsilyl)thymidine (3, 1.85 g, 5 mmol),3'-O-(t-butyldimethylsilyl)thymidine-5'-aldshyde [5, 2.39 g, 5 mmol;freshly prepared by following the method of Camarasa, et al.,Nucleosides and Nucleotides 1990, 9, 533] and ACOH (0.25 ml) werestirred together in CH₂ Cl₂ (50 ml) solution at room temperature for 2h. The products were then concentrated under reduced pressure to givethe intermediate oxime linked dimer, compound 6.

Step 2

The residue obtained from Step 1 was dissolved in AcOH (25 ml). NaCNBH₃(1.55 g, 25 mmol, in 3-portions) was added to the stirred AcOH solutionat room temperature. The solution was stirred for 30 min to give theintermediate imine linked dimer, compound 7.

Step 3

Aqueous HCHO (20%, 2 ml, 66 mmol) and additional NaCNBH₃ (1.55 g, 25mmol, in 3-portions) was added to the stirred reaction mixture of Step 2at room temperature. After 2 h, the solution was diluted with EtOH (100ml), and resulting suspension was evaporated under reduced pressure. Theresidue was dissolved in CH₂ Cl₂ (150 ml) and then washed successivelywith 0.1 M HCl (100 ml), saturated aqueous NaHCO₃ (100 ml), and water(2-50 ml). The dried (MgSO₄) CH₂ Cl₂ solution was evaporated to givecrude methylated imine linked dimer 8.

Step 4

The residue from Step 3 was dissolved in the THF (30 ml) and a solutionof (Bu)₄ NF (1 M in THF, 10 ml) was added while stirring at roomtemperature. After 1 h, the reaction mixture was evaporated underreduced pressure and the residue was purified by short columnchromatography. The appropriate fractions, which eluted with CH₂ Cl₂:MeOH (8:2, v/v) were pooled and evaporated to give compound 9 as a foam(0.74 g, 30%). ¹ H NMR (Me₂ SO-d₆) δ 1.78 (s, 6, 2CH₃), 2.10 (m, 4,2'CH₂), 2.5 (s, 3, N--CH₃), 2.8 (m, 2, 5'-N--CH₂), 3.6-4.08 (5m, 6,5'CH₂, 4' CH, 3' CH), 4.75 and 5.3 (2 br s, 2, 3' and 5' OH), 6.02 (d,1, 1'H), 6.1 (t, 1, 1'H), 7.4 and 7.45 (2s, 2, 2C₆ H), 11.3 (br s, 2,NH).

EXAMPLE 9

5'-O-(t-Butyldimethylsilyl)-3'-Deoxy-3'-[(Methyleneamino)-oxy]thymidine,10

A solution of HCHO (20% aqueous, 1 ml) was added dropwise to a stirredsolution of 3'-O-amino-5'-O-(t-butyldimethylsilyl)thymidine (3, 7.42 g,20 mmol) in dry MeOH (400 ml) at room temperature. After 6 h, anotherportion of HCHO (20% aqueous, 1.5 ml) was added and stirring continuedfor 16 h. The resulting solution was evaporated under reduced pressure,and the residue was purified by chromatography on silica gel to givecompound 10 (7.25 g, 95%) as clear foam. ¹ H NMR (CDCl₃) δ 0.1 [s, 3,(CH₃)₂ ], 0.9 [s, 9, (CH₃)₃ ], 1.9 (s, 3, CH₃), 2.25-2.72 (m, 2, 2'CH₂), 3.85-4.15 (2m, 3, 5' CH₂, 4' H), 4.85 (m, 1, 3'H), 6.25 (dd, 1,1'H), 6.5 and 6.95 (2d, 2, N═CH₂), 7.43 (s, 1, (6H), 9.2 (br s, 1 NH).

EXAMPLE 10

3'-O-(t-Butyldiphenylgilyl)-5'-Deoxy-5'-Iodothymidine 12

To a stirred solution of 3'-O-(t-butyldiphenylsilyl)thymidine [11, 10.0g, 20.83 mmol, prepared according to the procedure of Koster, et al.,Tet. Letts. 1982, 26, 2641] in dry DMF (375 ml) was addedmethyltriphenoxyphosphonium iodide (12.12 g, 30 mmol) under argon atroom temperature. The solution was stirred for 16 h. The DMF was removedunder reduced pressure and the residue was dissolved in CH₂ Cl₂ (500ml). The organic layer was washed with 20% aqueous Na₂ S₂ O₃ (200 ml),water (2×200 ml) and dried (MgSd₄). The solvent was evaporated and theresidue was purified by silica gel chromatography. Elution with Et₂ O:Hexanes (1:1, v/v), pooling of appropriate fractions and concentrationfurnished compound 12 as white power (7.87 g, 64%, mp 142° C.). Anal.Calcd. for C₂₆ H₃₁ N₂ O₄ SiI: C, 52.88; H, 5.29; N., 4.74; I, 21.33.Found: C,52.86; H, 5.21; N, 4.66; I, 21.54.

EXAMPLE 11

5'-O-(t-Butyldinothylsilyl)-3'-O-Dephouphinico-3'-O-(Imino-methylene)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,13

A stirred solution of5'-O-(t-butyldimethylsilyl)-3'-deoxy-3'-[(methyleneamino)oxy]thymidine(10, 1.62 g, 4.23 mmol),3'-O-(t-butyldiphenylsilyl)-5'-deoxy-5'-iodothymidine (12, 2.5 g, 4.23imol), bis(trimethylstannyl)benzopinacolate [4.84 g, 8.46 mmol, preparedaccording to the method of Hillgartner, et al., Liebigs Ann. Chem. 1975,586] in dry benzene (9 ml) was carefully degassed 3-times (flushed withargon) and heated at 80° C. for 8 h. The reaction mixture was cooled andconcentrated under reduced pressure and the residue was purified bysilica gel chromatography. The appropriate fractions, which were elutedwith CH₂ Cl₂ :MeOH (97:3, v/v), were pooled and concentrated to givedimeric oligonucleoside, compound 13 (1.25 g, 35%) as white foam. ¹ HNMR (CDCl₃) δ 0.09 and 0.13 [2s, 6, (CH₃)₂ ], 0.89 and 1.06 [2s, 9,(CH₃)₃ ], 1.07 and 1.08 [2s, 9, (CH₃)₃ ], 1.87, and 1.90 (2s, 6, 2 CH₃),5.74 (br s, 1, NH), 6.20-6.31 (2m, 2, 2 1'H), 6.88 (s, 1, C₆ H), 10.33and 10.36 (2 br s, 2, 2NH) and other protons.

EXAMPLE 12

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thysidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine14

Method A

Compound 13 was treated as per the procedure of Step 3 of Example 8-tosimultaneously N-alkylate the imino nitrogen and deblock the 5' silylblocking group of the 5' nucleoside of the dimer to yield compound 14 asa foam. ¹ H NMR (CDCl₃) δ 1.07 (9, 9, (CH₃)₃), 1.85 and 1.88 (2s, 6,2CH₃), 2.56 (8, 3, N--CH₃), 4.77 (br s, 1, 5' OH), 6.1 and 6.2 (2m, 2,1'H), 7.4 and 7.62 (2m, 10, Ph H), 9.05 (br s, 2, 2 NH), and otherprotons.

EXAMPLE 13

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-5'-Deoxythymidine,15

The 3'-O-(t-butyldiphenylsilyl) blocking group of compound 14 is removedas per the procedure of Step 4 of Example 8 to yield the fully deblockeddimeric oligo-nucleoside, compound 15.

EXAMPLE 14

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]-5'-Iodo--5'-Deoxythymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deozythysidine,16

Compound 14 is treated as per the procedure of Example 10 to yield thetitle dimeric oligonucleoside, compound 16, having a reactive iodofunctionality at the terminal 5' position and a blocking group remainingat the 3' position.

EXAMPLE 15

5'-O-(t-Butyldinethylsilyl)-3'-O-Dephosphinico-3'-O-(Imino-methylene)thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-[(-Mthyimino)methylene]-5'-Deozythymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deozythymidine,17

Compound 16 is reacted with compound 10 utilizing the conditions ofExample 11 to extend the oligonucleoside to yield the trimericoligonucleoside, compound 17.

EXAMPLE 16

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-5'-Deoxythymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidinc,18

Compound 17 when reacted as per the conditions of Example 12 willundergo N-alkylation to the trimeric oligonucleoside and will be deblockat the 5' position to yield compound 18, wherein n=2.

EXAMPLE 17

3'-O-Dophosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-5'-Deozythymidylyl-(3'.fwdarw.5')-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-5'-Deozythymidylyl-(3'→5')-5'-Deozythymidine,20

The sequence of Examples 13, 14, and 15 is repeated for the addition ofa further nucleoside to extend the oligonucleoside to a tetramer,compound 19. The tetrameric oligonucleoside 19 is then treated as perthe procedure of Example 13 to remove the terminal 3' silyl blockinggroup yielding the fully deblocked tetrameric oligonucleoside, compound2.

EXAMPLE 18

5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'[(Methyleneamino)oxy]adenosine, 27;5'-O-(t-Butyldiaethylsilyl)-2',3'-Dideozy-3'-[(Methylaneamino)oxy]cytidine,28; and5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'-[(Methyleneamino)oxy]guanosine,29

3'-O-Amino-2'-deoxyadenosine, compound 24, 3'-O-amino-2'-deoxycytidine,compound 25, and 3'-O-amino-2'-deoxyguanosine, compound 26, prepared asper the procedures of European Patent Application 0 381 335 or in amanner analogous to the preparation of compound 4 by the procedure ofExample 7 above, are blocked at their 5' position with at-butyldimethylsilyl group according to the procedure of Nair, et al.,Org. Prep. Procedures Int. 1990, 22, 57 to give the corresponding3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyadenosine,3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxycytidine and3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyguanosine nucleosideinter-mediates. Treatment of the blocked intermediate as per theprocedure of Example 9 or as per the procedure of Preparation Example 4of European Patent Application 0 381 335 gives the corresponding5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]adenosine,compound 27;5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]cytidine,compound 28; and5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methylene-amino)oxy]guanosine,compound 29.

EXAMPLE 19

Dimer Synthesis

5'-Benzoyl-3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythysidine,35

Compound 34 is reacted with silylated thymine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene to yield5'-O-benzoyl-3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxythymidine,compound 35 as an anomeric mixture.

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]-thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-adenosine,36

Compound 34 is reacted with silylated adenine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)-methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyadenosine,36.

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene-]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxycytidine37

Compound 34 is reacted with silylated cytosine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)-methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxycytidine,37.

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]-thymidylyl-(3'→5')-3'-O-(t-Butyldiphenymlsilyl)-5'-Deoxyguanosin38

Compound 34 is reacted with silylated guanine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)-methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyguanosine,38.

A-(3'→5')-T; A-(3'→5')-A; A-(3'→5')-C; and A-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)Methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoadenosine intermediate of Example 15will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is adenine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the A-T, A-A, A-C and A-G diners,respectively, of a structure equivalent to that of compound 14 where Bxiis adenine and Bxj is thymine, adenine, cytosine and guanine,respectively.

C-(3'→5')-T; C-(3'→5')-A; C-(3'→5')-C; and C-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminocytidine intermediate of Example 15will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is cytidine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the C-T, C-A, C-C and C-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis cytosine and Bxj is thymine, adenine, cytosine and guanine,respectively.

G-(3'→5')-T; G-(3'→5')-A; G-(3'→5')-C; and G-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoguanosine intermediate of Example 15will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is guanine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the G-T, G-A, G-C and G-G diners,respectively, of a structure equivalent to that of compound 14 where Bxiis guanine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 20

Trimeric, Tetraueric, Pentameric, Hexameric And Other Higher OrderOligonucleosides Having a Selected Nucleoside sequence

The dimers of Examples 19 are extended by reaction with the5'-(t-butyldimethylsilyl)-3'-deoxy-3'-[(methyleneamino)oxy] nucleosides,compounds 10, 27, 28 and 29, of Examples 8 and 17 to form trimersutilizing the looping sequence of reactions of Examples 13, 14, and 15.Iteration of this reaction sequence loop adds a further nucleoside tothe growing oligonucleoside per each iteration of the reaction sequenceloop. The reaction sequence loop of Examples 13, 14, and 15 is repeated"n" number of times to extend the oligonucleoside to the desired "n+1"length. The final 3'-blocked oligonucleoside when treated as per theprocedure of Example 13 to remove the terminal3'-O-(t-butyldiphenylsilyl) blocking group will yield the fullydeblocked oligonucleoside of the selected nucleoside sequence andlength.

EXAMPLE 21

5'-O-(t-Butyldinethylsilyl)-3'-O-Dephosphinico-3'-O-(Iminomethylene)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilil)-5'-Deoxy-5'-Hydroxythymidine,48

Utilizing the procedure of Hanamoto, et al., Tet. Letts. 1991, 32, 3555,SmI₂ (0.1 mmol) in THF (3 ml) is added to a mixture of compound 5 andcompound 10 in HMPA (0.5 ml) with stirring. The mixture will be stirredat room temperature for about 15 mins to form the adduct (as detected bythe fading color). The solvent will be removed and the residue purifiedby column chromatography to give the dimeric oligonucleoside 48.

EXAMPLE 22

3'-O-Dephosphinico-3'-O-[N-(Morpholin-2-yl)]thymidylyl-(3'→4')-3'-O-(t-Butyldipheoylsilyl)-5'-Deoxy-5'-Demethylenethymidine,49

Utilizing the modification of Lim, M.-I. and Pan, Y.-G., Book ofAbstracts, 203 ACS national Meeting, San Francisco, Calif., Apr. 5-10,1992, of the procedure of Hill, et al., J. Chem. Soc. 1964, 3709, thedimeric oligonucleoside of Example 21 (compound 48, 1 equiv.) will betreated with chloroacetyl chloride in acetone to form an adduct with theamino group of the linkage. Further treatment with K₂ CO₃ (1.2 equiv.)in DMSO at elevated temperature will cyclize the adduct to the hydroxylgroup of the linkage to form a 5-oxomorpholino adduct with the linkage.The oxomorpholino adduct is then reduced with BH₃ -THF under reflux toyield the dimer linked via an --O--[N-(morpholin-2-yl)]-linkage,compound 49.

EXAMPLE 23

Synthesis Of Oligonucleotides Using A DNA Synthesizer

Solid support oligonucleotide and "oligonucleotide like" syntheses areperformed on an Applied Biosystems 380 B or 394 DNA synthesizerfollowing standard phosphoramidite protocols and cycles using reagentssupplied by the manufacture. The oligonucleotides are normallysynthesized in either a 10 μmol scale or a 3×1 μmol scale in the"Trityl-On" mode. Standard deprotection conditions (30% NH₄ OH, 55° C.,16 hr) are employed. HPLC is performed on a Waters 600E instrumentequipped with a model 991 detector. For analytical chromatography, thefollowing reverse phase HPLC conditions are employed: Hamilton PRP-1column (15×2.5 cm); solvent A: 50 mm TEAA, pH 7.0; solvent B: 45 mm TEAAwith 80% CH₃ CN; flow rate: 1.5 ml/min; gradient: 5% B for the first 5minutes, linear (1%) increase in B every minute thereafter. Forpreparative purposes, the following reverse phase HPLC conditions areemployed: Waters Delta Pak Waters Delta-Pak C₄ 15 μm, 300A, 25×100 mmcolumn equipped with a guard column of the same material; column flowrate: 5 ml/min; gradient: 5% B for the first 10 minutes, linear 1%increase for every minute thereafter. Following HPLC purification,oligonucleotides are detritylated and further purified by size exclusionusing a Sephadex G-25 column.

EXAMPLE 24

Higher Order Mixed Oligonucleosides-oligonucleosides And Mixedoligonucleosides-oligonucleotides

A. Solution Phase Synthesis Of3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylone)]-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-3'-Phosphorothioate-Thymidylyl-(3'→5')-3'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,90, A Mixed oligonucleoside-oligonualeotide-oligonucleoside PolymerIncorporating A Nucleotide Linkage Flanked At Its 5' Terminus By A3'-De(oxophosphinico)-3'-[Methyl(iminoozymethylene)] LinkedOligonucleoside Dimer and At Its 3' Terminus By A3'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-Ethanediyl] Linkedoligonucleoside Dimer

A mixed oligonucleoside-oligonucleotide-oligonucleotside having a3'-de(oxophosphinico)-3'-[methyl(iminooxymethylene)]linkedoligonucleoside dimer and a3'-de(oxyphosphinico)-3"-[(methylimino)-1,2-ethanediyl] linkedoligonucleoside dimer coupled together via a phosphorothioate nucleotidelinkage will be prepared by reacting compoundy 58, compound 70 andtetrazole in anhydrous acetonitrile under argon. The coupling reactionwill be allowed to proceed to completion followed by treatment withBeaucage reagent and ammonium hydroxide removal of the dimethoxytritylblocking group according to the procedure of Zon, G. and Stec, W. J.,Phosphorothioate oligonucleotides, oligonucleotides and Analogs APractical Approach, F. Eckstein Ed., IRL Press, pg. 87 (1991). The 3'blocking group will then removed as per the procedure of Step 3 ofExample 8 and the product purified by HPLC to yield the title compound90, wherein utilizing the structure of Scheme XI, T₃ and T₅ are OH, D isS, E is OH, X is H, Q is O, r is O and q is 2; and for each q, i.e. q₁and q₂, n and p are 1 in each instance; and for q₁, m is 1; and for q₂,m is 0; and Bxj and Bxi are thymine.

B. Solid Support Synthesis Of3'-De(oxophosphinico)-3"-[Methyl(iminoozymethylone)]-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-(3'→5')-P-Thylidylyl-3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylone)]-(3'→5')-Thymidylyl-(3'→5')-P-Thylidylyl-3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-(3'→5')-Thymidylyl-(3'→5')-P-2'-Deoxycytidine,91, A Mized Oligonucleotide-Oligonuclooside Polymer incorporating3'-De(oxophos-phinico)-3'-[Methyl(iminooxymthylemn)] LinkedOligonucleoside Dimers Planked By Conventional Linked Nuclcotides

The dimeric oligonucleoside 58 will be utilized as building block unitsin a conventional oligonucleotide solid support synthesis as per theprocedure of Example 23. For the purpose of illustration a polymerincorporating seven nucleosides is described. A first unit of thedimeric oligonucleoside 58 will be coupled to a first cytidinenucleoside tethered to a solid support via its 3' hydroxyl group andhaving a free 5' hydroxyl group. After attachment of the first unit ofcompound 58 to the support, the 5'-dimethoxytrityl group of that firstcompound 58 unit will be removed in the normal manner. A second compound58 unit will then be coupled via itsβ-cyanoethyl-N-diisopropylphosphiryl group to the first compound 58 unitusing normal phosphoramidate chemistry. This forms a conventionalphosphodiester bond between the first and second compound 58 units andelongates the polymer by two nucleosides (or one oligonucleoside dimerunit). The dimethoxytrityl blocking group from the second compound 58unit will be removed in the normal manner and the polymer elongated by afurther dimeric unit of compound 58. As with addition of the first andsecond dimeric units, the third unit of compound 58 is coupled to thesecond via conventional phosphoramidite procedures. The addition of thethird unit of compound n completes the desired length and base sequence.This polymer has a backbone of alternating normal phosphodiesterlinkages and the methyl-(iminooxymethylene) linkages of compound 58. The5' terminal dimethoxytrityl group of the third compound 58 unit will beremoved in the normal manner followed by release of the polymer from thesolid support, also in the normal manner. Purification of the polymerwill be achieved by HPLC to yield compound 91 wherein, utilizing thestructure of Scheme XI, T₃ and T₅ are OH, D is O, E is OH, X is H, Q isO, r is 1 and for the seven nucleoside polymer described, q is 3; andfor each q, i.e. q₁, q₂ and q₃, n and p are 1 in each instances; and forq₁ and q₂, m is 1; and for q₃, n is 0; and Bxk is cytosine; and each BxJand Bxi is thymine.

EXAMPLE 25

General method for 3'-deoxy-3'-iodo nucleosides

The preparation of 3'-deoxy-3'-iodo-5'-O-tritylthymidine has beendescribed by Verhyden, et al., J. Org. Chem. 1970, 35, 2868. In ananalogous manner, 2',3'-dideoxy-3'-iodo-5'-O-trityluridine, cytidine,adenosine and guanosine will be prepared.

EXAMPLE 26

Synthesis of Bifunctional Nucleosides

5"-Deoxy-5'iodo-3'-O-phthalisidothysidine, 102

Treatment of 3'-O-phthalimidothymidine with methyltriphenoxyphosphoniumiodide (Example 10) furnished 48% of 102; m.p. 145-146° C.; Anal. Calcd.for C₁₈ H₁₆ N₃ O₆ I: C, 43.48; H, 3.24; N, 8.48; I, 25.52. Found: C,43.75; H, 3.34; N, 8.38; I, 25.59. ¹ H NMR (CDCl₃) δ 8.01 (s, 3, C₅CH₃), 2.58-2.67 (m, 2, 2'CH₂), 3.54-3.78 (m, 2, 5' CH₂), 4.30-4.34 (m,1, 4'H), 5.01-5.03 (m, 1, 3'H), 6.38 (dd, 1, 1'H), 7.77-7.78 (m, 6, C₅ Hand ArH), 8.69 (br s, 1, NH).

3'-deozy-3'-iodo-5'-O-phthalimidothymidine, 109

Treatment of 5'-O-phthalimidothymidine (Example 1) withmethyltriphenoxyphosphoniuniodide in an analogous manner gave 43% of 1;m.p. 130 (decomposes); Anal. Calcd. for C₁₈ H₁₆ N₃ O₆ I:C, 43.75; H,3.24; N, 8.45; I, 25.52. Found: C, 54.82, H, 3.24; N, 8.45; I, 25.18. ¹H NMR (CDCl₃) δ 1.94 (s, 3, C5CH₃), 2.70-2.79 (m, 2, 2'CH₂), 4.53-4.56(m, 3, 5'CH₂, 3'H), 4.67 (m, 1, 4'H), 6.28 (5, 1, 1'H), 7.70 (5, 1, CH₆H), 7.71-7.90 (m, 4, ArH), 8.55 (br s, 1, NH).

EXAMPLE 27

Incorporation Of Phosphodiester Linkages

Dimeric nucleosides 117c (R'=ODMTr, R"=O-amidite) and 119b (R'=ODMTr,R"=O-amidite), and trimeric nucleoside 118d (R'=ODMTr, R"=O-amidite, L₂=L_(2a) =N--CH₃) were prepared generally in accordance with Sproat, etal., Oligonucleotides and Analogs A Practical Approach, Eckstein, ed.,IRL Press, 1991.

3'-De(oxyphosphinico)-3'-O-(iminomethylene)-5'-dimethyoxytritylthymidylyl-(3'→5')-3"-[(β-cyanoethyoxy)-N-(diisopropyl)phosphiryl]-5'-deoxythymidine117c was obtained as a white foam (mixture of diastereoisomer): ³¹ P NMR(CD₃ CN) δ 149.1 and 149.5 ppm; ¹ H NMR (CD₃ CN) δ 1.6 and 1.75 (2S, 62C₅ CH₃), 2.20 (S, 3, N--CH₃), 6.1 (m, 2, 1'H), 9.0 (br S, 2, NH) andother protons.

3'-De(oxyphosphinico)-3'-[(methylene(methylimino)]-thymidylyl-5'-O-(dimethytrityl)-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphilyl)thymidine 118d was obtained as whiteproipitate (mixture of diastereoisomer): 31P NMR (CDCl₃) δ 149.62 and149.11 ppm; ¹ H NMR (CDCl₃) δ 1.82 and 1.49 (2S, 6, 2C₅ CH₃), 2.58 and2.56 (2S, 3, N--CH₃), 6.16 (pseudo t, 1, T1-1'H, J=_(1'),2' =J_(1'),2"=5.8 Hz), 6.22 (pseudo t, 1, T2 1'H, J=_(1'),2' =J_(1'),2" =6.7 Hz), andother protons.

Phosphoramidites 117c, 118d, and 119b can be stored and used forcoupling by automated DNA synthesizer (e.g., ABI 380 B) as and whenrequired for specific incorporation into oligomers of therapeutic value.Other dimers of the inventions can be incorporated into oligomers in asimilar manner. This permits flexibility in converting oligonucleosidesprepared via radical coupling methodology of this invention intostandard phosphoramidites, which can be utilized as "blocks of syntheticDNA" to improve the pharmacokinetic and pharmacodynamic properties ofantisense oligomers.

EXAMPLE 28

Enzymatic Degradation

5'GCGTTTTT*TTTTTGCG3' (*=3'--CH₂ --N(CH₃)--O--CH₂ --4' linkage; 30nanomoles) was dissolved in 20 ml of buffer containing 50 mM Tris-HCl pH8.5, 14 mM MgCl₂, and 72 mM NaCl. To this solution 0.1 units ofsnake-venom phosphodiesterase (Pharmacia, Piscataway, N.J.), 23 units ofnuclease P1 (Gibco LBRL, Gaithersberg, Md.), and 24 units of calfintestinal phosphatase (Boehringer Mannheim, Indianapolis, Ind.) wasadded and the reaction mixture was incubated at 37° C. for 100 h. HPLCanalysis was carried out using a Waters model 715 automatic injector,model 600E pump, model 991 detector, and an Alltech (Alltech Associates,Inc., Deerfield, Ill.) nucleoside/nucleotide column (4.6×250 mm). Allanalyses were performed at room temperature. The solvents used were A:water and B: acetonitrile. Analysis of the nucleoside composition wasaccomplished with the following gradient: 0-5 min., 2% B (isocratic);5-20 min., 2% B to 10% B (linear); 20-40 min., 10% B to 50% B. Theintegrated area per nanomole was determined using nucleoside standards.The T*T diner containing the N-methylaminohydroxy linkage wasquantitated by evaluation as if it were a thymidine nucleoside. Relativenucleoside ratios were calculated by converting integrated areas tomolar values and comparing all values to thymidine, which was set at itsexpected value for each oligomer.

EVALUATION

PROCEDURE 1-Nuclease Resistance

A. Evaluation of the resistance of oligonucleotide-mimickinqmacromolecules to serum and cytoplasmic nucleases.

Oligonucleotide-mimicking macromolecules of the invention can beassessed for their resistance to serum nucleases by incubation of theoligonucleotide-mimicking macromolecules in media containing variousconcentrations of fetal calf serum or adult human serum. Labeledoligonucleotide-mimicking macromolecules are incubated for varioustimes, treated with protease K and then analyzed by gel electrophoresison 20% polyacrylamine-urea denaturing gels and subsequentautoradiography. Autoradiograms are quantitated by laser densitometry.Based upon the location of the modified linkage and the known length ofthe oligonucleotide-mimicking macromolecules it is possible to determinethe effect on nuclease degradation by the particular modification. Forthe cytoplasmic nucleases, an HL 60 cell line can be used. Apost-mitochondrial supernatant is prepared by differentialcentrifugation and the labelled macromolecules are incubated in thissupernatant for various times. Following the incubation, macromoleculesare assessed for degradation as outlined above for serum nucleolyticdegradation. Autoradiography results are quantitated for evaluation ofthe macromolecules of the invention. It is expected that themacromolecules will be completely resistant to serum and cytoplasmicnucleases.

B. Evaluation of the resistance of oligonucleotide-simickingmacromolecules to specific endo- and ezo-nucleases.

Evaluation of the resistance of natural oligonucleotides andoligonucleotide-mimicking macromolecules of the invention to specificnucleases (ie, endonucleases, 3',5'-exo-, and 5',3'-exonucleases) can bedone to determine the exact effect of the macromolecule linkage ondegradation. The oligonucleotide-mimicking macromolecules are incubatedin defined reaction buffers specific for various selected nucleases.Following treatment of the products with protease K, urea is added andanalysis on 20% polyacrylamide gels containing urea is done. Gelproducts are visualized by staining with Stains All reagent (SigmaChemical Co.). Laser densitometry is used to quantitate the extent ofdegradation. The effects of the macromolecules linkage are determinedfor specific nucleases and compared with the results obtained from theserum and cytoplasmic systems. As with the serum and cytoplasmicnucleases, it is expected that the oligonucleotide-mimickingmacromolecules of the invention will be completely resistant to endo-and exo-nucleases.

C. Nuclease Degradation studies

It has been reported that terminal phosphorothioate andmethylphosphonate modifications stabilize an oligonucleotide to 3' and5' exonucleases such as snake venom phosphodiesteraser, spleenphosphodiesterase, calf serum, and cell media (see, e.g., Nucleic AcidsRes. 1991, 19, 747 and 5473). The novel backbones of this inventionconfer similar or sometimes better protection from enzymaticexonucleolytic degradation. The inventors recently reported (J. Am.Chem. Soc. 1992, 114, 4006) that incorporation of a single 3'-CH₂--N(CH₃)--O--CH2--4' backbone at the 3' terminus of an oligomer enhancedthe half-life of modified oligomer compared to the natural unmodifiedoligomer. These oligonucleosides exhibited a significant resistance tonucleases while maintaining a high level of base pair specificity.Therefore, the following oligonucleosides were modified at theirterminal positions (3' and/or 5') with 3'--CH₂ --N(CH₃)--O--CH2--4'linkages to block exonucleolytic degradation.

    ______________________________________                                                Oligonucleoside                                                                              No. Sequence T 1/2 (Hours)                             ______________________________________                                        1       TTTTTTTTTTC   0.2                                                       2 T*TT*TTTT*TT*TC 0.4 (11 mer) 11 (10-mer)                                    3 T*TTTTTTTT*TC 0.4 (11-mer) 6 (10-mer)                                       4 T*TT*TT*TT*TT*TC 0.4 (11-mer) 16 (10-mer)                                   5 T*T*T*T No degradatian!                                                       (up to ˜ 60 hours)                                                  ______________________________________                                    

Results for Entries 1-4, above, correspond to SEQ ID NO: 1 Entries 2-4showed a characteristic 3'-exonuclease-dominant degradation patterncharacterized by rapid cleavage of 3'-C residue from all oligomers.Aside from the 3'-phosphodiester linkages, the 10-mers appear to besignificantly resistant towards degradation compared to the unmodifiedoligomer (Entry 1). The fully modified tetramer (Entry 5), whichcontains no phosphodiester linkage, showed complete stability up to 60hours of incubation in cell extract (see, Nucleic Acids Res. 1991, 19,5743 for experimental details). Since the phosphodiester linkage is thesite of nucleolytic attack, complete stability for the fully modifiedoligomer was expected. These results taken together suggests that anend-capped (3' and 5') oligomer containing achiral and neutral backbonewill have enhance half-life.

PROCEDURE 2-5-Lipoxygenase Analysis and Assays

A. Therapeutics

For therapeutic use, an animal suspected of having a diseasecharacterized by excessive or abnormal supply of 5-lipoxygenase istreated by administering the macromolecule of the invention. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Such treatment is generallycontinued until either a cure is effected or a diminution in thediseased state is achieved. Long term treatment is likely for somediseases.

B. Research Reagents

The oligonucleotide-mimicking macromolecules of this invention will alsobe useful as research reagents when used to cleave or otherwise modulate5-lipoxygenase mRNA in crude cell lysates or in partially purified orwholly purified RNA preparations. This application of the invention isaccomplished, for example, by lysing cells by standard methods,optimally extracting the RNA and then treating it with a composition atconcentrations ranging, for instance, from about 100 to about 500 ng per10 Mg of total RNA in a buffer consisting, for example, of 50 mmphosphate, pH ranging from about 4-10 at a temperature from about 300 toabout 50° C. The cleaved 5-lipoxygenase RNA can be analyzed by agarosegel electrophoresis and hybridization with radiolabeled DNA probes or byother standard methods.

C. Diagnostics

The oligonucleotide-mimicking macromolecules of the invention will alsobe useful in diagnostic applications, particularly for the determinationof the expression of specific mRNA species in various tissues or theexpression of abnormal or mutant RNA species. In this example, while themacromolecules target a abnormal mRNA by being designed complementary tothe abnormal sequence, they would not hybridize to normal mRNA.

Tissue samples can be homogenized, and RNA extracted by standardmethods. The crude homogenate or extract can be treated for example toeffect cleavage of the target RNA. The product can then be hybridized toa solid support which contains a bound oligonucleotide complementary toa region on the 5' side of the cleavage site. Both the normal andabnormal 5' region of the mRNA would bind to the solid support. The 3'region of the abnormal RNA, which is cleaved, would not be bound to thesupport and therefore would be separated from the normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however,persons of ordinary skill in the art will appreciate that the presentinvention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

D. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of oligonucleotide-mimicking macromolecules whichinterfere with 5-lipoxygenase synthesis in these cells.

A second test system for oligonucleotide-mimicking macromolecules makesuse of the fact that 5-lipoxygenase is a "suicide" enzyme in that itinactivates itself upon reacting with substrate. Treatment ofdifferentiated HL-60 or other cells expressing 5 lipoxygenase, with 10μM A23187, a calcium ionophore, promotes translocation of 5-lipoxygenasefrom the cytosol to the membrane with subsequent activation of theenzyme. Following activation and several rounds of catalysis, the enzymebecomes catalytically inactive. Thus, treatment of the cells withcalcium ionophore inactivates endogenous 5-lipoxygenase. It takes thecells approximately 24 hours to recover from A23187 treatment asmeasured by their ability to synthesize leukotriene B. Macromoleculesdirected against 5-lipoxygenase can be tested for activity in two HL-60model systems using the following quantitative assays. The assays aredescribed from the most direct measurement of inhibition of5-lipoxygenase protein synthesis in intact cells to more downstreamevents such as measurement of 5-lipoxygenase activity in intact cells.

A direct effect which oligonucleotide-mimicking macromolecules can exerton intact cells and which can be easily be quantitated is specificinhibition of 5-lipoxy-genase protein synthesis. To perform thistechnique, cells can be labelled with ³⁵ S-methionine (50 μCi/mL) for 2hours at 37° C. to label newly synthesized protein. Cells are extractedto solubilize total cellular proteins and 5-lipoxygenase isimmunoprecipitated with 5-lipoxygenase antibody followed by elution fromprotein A Sepharose beads. The immunoprecipitated proteins are resolvedby SDS-polyacrylamide gel electrophoresis and exposed forautoradiography. The amount of immunoprecipitated 5-lipoxygenase isquantitated by scanning densitometry.

A predicted result from these experiments would be as follows. Theamount of 5-lipoxygenase protein immunoprecipitated from control cellswould be normalized to 100%. Treatment of the cells with 1 μM, 10 μM,and 30 μM of the macromolecules of the invention for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control,respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenatescould also be used to quantitate the amount of enzyme present which iscapable of synthesizing leukotrienes. A radiometric assay has now beendeveloped for guantitating 5-lipoxygenase enzyme activity in cellhomogenates using reverse phase HPLC. Cells are broken by sonication ina buffer containing protease inhibitors and EDTA. The cell homogenate iscentrifuged at 10,000×g for 30 min and the supernatants analyzed for5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM ¹⁴C-arachidonic acid, 2 mM ATP, 50 μM free calcium, 100 μg/mlphosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37°C. The reactions are quenched by the addition of an equal volume ofacetone and the fatty acids extracted with ethyl acetate. The substrateand reaction products are separated by reverse phase HPLC on a NovapakC18 column (Waters Inc., Millford, Mass.). Radioactive peaks aredetected by a Beckman model 171 radiochromatography detector. The amountof arachidonic acid converted into di-HETE's and mono-HETE's is used asa measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for72 hours with effective the macromolecules of the invention at 1 μM, 10μM, and 30 μM would be as follows. Control cells oxidize 200 pmolarachidonic acid/5 min/106 cells. Cells treated with 1 μM, 10 μM, and 30μM of an effective oligonucleotide-mimicking macromolecule would oxidize195 pmol, 140 pmol, and 60 pmol of arachidonic acid/5 min/10⁶ cellsrespectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) forthe measurement of total 5-lipoxygenase protein in cells has beendeveloped. Human 5-lipoxygenase expressed in E. coli and purified byextraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is usedas a standard and as the primary antigen to coat microtiter plates. 25ng of purified 5-lipoxygenase is bound to the microtiter platesovernight at 4° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM TrisHCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labelled secondantibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30 meroligonucleotide-mimicking macromolecule at 1 μM, 10 μM, and 30 μM wouldbe 30 ng, 18 ng and 5 ng of 5-lipoxygenase per 10⁶ cells, respectivelywith untreated cells containing about 34 ng 5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophil-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of the macromolecule for 48-72 hours in thepresence of 1.3% DMSO. The cells are washed and resuspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining 1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

Using this assay the following results would likely be obtained with anoligonucleotide-mimicking macromolecule directed to the 5-LO mRNA. Cellswill be treated for 72 hours with either 1 μM, 10 μM or 30 μM of themacromolecule in the presence of 1.3% DMSO. The quantity of LTB₄produced from 5×10 cells would be expected to be about 75 pg, 50 pg, and35 pg, respectively with untreated differentiated cells producing 75 pgLTB₄.

E. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can bedemonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oigonucleotide-mimicking macro-molecules will be appliedtopically to both ears 12 to 24 hours prior to administration ofarachidonic acid to allow optimal activity of the compounds. Both earsare pre-treated for 24 hours with either 0.1 μmol, 0.3 μmol, or 1.0 μmolof the macromolecule prior to challenge with arachidonic acid. Valuesare expressed as the mean for three animals per concentration.Inhibition of polymorphonuclear cell infiltration for 0.1 μmol, 0.3μmol, and 1 μmol is expected to be about 10%, 75% and 92% of controlactivity, respectively. Inhibition of edema is expected to be about 3%,58% and 90%, respectively while inhibition of leukotriene B₄ productionwould be expected to be about 15%, 79% and 99%, respectively.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such-equivalent variations as fall within thetrue spirit and scope of the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 1                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (iv) ANTI-SENSE:  No                                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 1:                         - - TTTTTTTTTT C               - #                  - #                      - #       11                                                                __________________________________________________________________________

What is claimed:
 1. A compound having structure: ##STR8## wherein: Z₁ isCH₂ --R_(B), R_(B), CH₂ --R_(A) N═CH₂, or R_(A) N═CH₂, provided that Z₁is not R_(A) N═CH₂ when X₁ is H or OH, and Z₁ is not R_(B) when R_(B) isOC(O)O--C₆ H₅ ;R_(A) is O or NH; R_(B) is a radical generating groupselected from I, OC(O)O--C₆ H₅, OC(O)S--C₆ H₅, Se--C₆ H₅, O--C(S)O--C₆F₅, O--C(S)O--C₆ Cl₅, O--C(S)O--(2,4,6--C₆ Cl₃), NO₂, C(S)S--Me,C(S)O--(p--CH₄ F), bis-dimethylglyoximato-pyridine cobalt, O--C(S)C₆ H₅,O--C(S)SCH₃, O--C(S)-imidazole, and C(O)O-pyridin-2-thione; Y₁ is H,hydroxymethyl, a nucleoside, a nucleotide, an oligonucleotide, anoligonucleoside, or a hydroxyl-protected or amine-protected derivativethereof; B_(X1) is a nucleosidic base; Q₁ is O, S, CH₂, CHF or CF₂ ; andX₁ is H, OH, C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl.
 2. The compound of claim 1 wherein R_(A) is O.
 3. Thecompound of claim 1 wherein R_(A) is NH.
 4. The compound of claim 1wherein Z₁ is CH₂ --R_(S) or R_(S).
 5. The compound of claim 1 whereinZ₁ is R_(A) --N═CH₂ or CH₂ --R_(a) -N═CH₂.
 6. The compound of claim 1wherein said radical generating group is I.
 7. A compound havingstructure: ##STR9## wherein: Y₂ is CH₂ --R_(B), R_(B), CH₂ --R_(A)N═CH₂, or R_(A) N═CH₂ ;R_(A) is O or NH; R_(B) is a radical generatinggroup selected from I, OC(O)O--C₆ H₅, OC(O)S--C₆ H₅, Se--C₆ H₅,O--(S)O--C₆ F₅, O--(S)O--C₆ Cl₅, O--(S)O--(2,4,6--C₆ Cl₃), Br, NO₂, Cl,C(S)S--Me, C(S)O--(p--CH₄ F), bis-dimethylglyoximato-pyridine cobalt,O(S)C₆ H₅, O(S)SCH₃, O(S)-imidazole, and C(O)O-pyridin-2-thione; Z₂ isH, hydroxyl, a nucleoside, a nucleotide, an oligonucleotide, anoligonucleoside, or a hydroxyl-protected or amine-protected derivativethereof; B_(X2) is a nucleosidic base; Q₂ is O, S, CH₂, CHF or CF₂ ; andX₂ is H, OH, C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl; provided that R_(B) is not I, Br, or Cl when Z₂ ishydroxyl, Q₂ is O, and X₂ is H or OH.
 8. The compound of claim 7 whereinR_(A) is O.
 9. The compound of claim 7 wherein R_(A) is NH.
 10. Thecompound of claim 7 wherein Y₂ is CH₂ --R_(S) or R_(S).
 11. The compoundof claim 7 wherein Y₂ is CH₂ --R_(A) --N═CH₂ or R_(A) --N═CH₂.
 12. Thecompound of claim 7 wherein said radical generating group is I.